Development of Mechanical Stability in Late-Stage Embryonic Erythroid Cells: Insights From Fluorescence Imaged Micro-Deformation Studies

The combined use of fluorescence labeling and micro-manipulation of red blood cells has proven to be a powerful tool for understanding and characterizing fundamental mechanisms underlying the mechanical behavior of cells. Here we used this approach to study the development of the membrane-associated cytoskeleton (MAS) in primary embryonic erythroid cells. Erythropoiesis comes in two forms in the mammalian embryo, primitive and definitive, characterized by intra- and extra-vascular maturation, respectively. Primitive erythroid precursors in the murine embryo first begin to circulate at embryonic day (E) 8.25 and mature as a semi-synchronous cohort before enucleating between E12.5 and E16.5. Previously, we determined that the major components of the MAS become localized to the membrane between E10.5 and E12.5, and that this localization is associated with an increase in membrane mechanical stability over this same period. The change in mechanical stability was reflected in the creation of MAS-free regions of the membrane at the tips of the projections formed when cells were aspirated into micropipettes. The tendency to form MAS-free regions decreases as primitive erythroid cells continue to mature through E14.5, at least 2 days after all detectable cytoskeletal components are localized to the membrane, indicating continued strengthening of membrane cohesion after membrane localization of cytoskeletal components. Here we demonstrate that the formation of MAS-free regions is the result of a mechanical failure within the MAS, and not the detachment of membrane bilayer from the MAS. Once a “hole” is formed in the MAS, the skeletal network contracts laterally along the aspirated projection to form the MAS-free region. In protein 4.1-null primitive erythroid cells, the tendency to form MAS-free regions is markedly enhanced. Of note, similar MAS-free regions were observed in maturing erythroid cells from human marrow, indicating that similar processes occur in definitive erythroid cells. We conclude that localization of cytoskeletal components to the cell membrane of mammalian erythroid cells during maturation is insufficient by itself to produce a mature MAS, but that subsequent processes are additionally required to strengthen intraskeletal interactions.

A lyophilized form of xenogeneic total RNA stimulates hematopoiesis in post-radiation myelosuppression

Введение. Аллогенная суммарная РНК, выделенная из клеток лимфоидных органов, стимулирует регенерацию кроветворной ткани после острого и хронического нарушения кроветворной функции. Цель исследования: 1) доказательство отсутствия ксеногенных ограничений механизмов лимфоцитарного контроля регенеративных процессов на примере гемостимулирующего действия суммарной РНК лимфоцитов бычьей селезенки в отношении кроветворения крыс, подвергшихся гамма-облучению в сублетальной дозе; 2) сравнительный анализ эффективности нативной и лиофилизированной форм указанной РНК. Методика. Работа выполнена на белых нелинейных крысах-самцах массой 200-220 г. Cуммарную РНК выделяли методом фенол - хлороформной экстракции из лимфоидных клеток бычьей селезенки. Для создания исходной миелосупрессии 30 крыс подвергли однократному общему воздействию гамма-излучения с источником 60Co в дозе 6 Гр при мощности дозы 0,1 Гр/с, после чего разделили их на 3 равные группы. Через 2 ч после облучения крысам контрольной группы внутрибрюшинно ввели по 0,5 мл 0,9% NaCl; крысам 2-й группы - нативную суммарную РНК в дозе 30 мкг/100г массы, крысам 3-й группы - лиофилизированную суммарную РНК в аналогичной дозе. На 3-и, 7-е и 12-е сут в периферической крови определяли количество ретикулоцитов, лейкоцитов и тромбоцитов, после чего крысы были выведены из эксперимента с целью исследования костномозгового кроветворения. Через 12 сут в костном мозге определяли количество эритроидных, лимфоидных, мегакариоцитарных и миелоидных клеток. Из костного мозга выделяли эритробластические островки (ЭО) и дифференцировали их на пролиферирующие (ЭО 1,2 классов и реконструирующиеся ЭО) и зрелые (ЭО 3 класса и инволюциирующие ЭО) морфо-функциональные клеточные ассоциации. Результаты. Под влиянием ксеногенной суммарной РНК в периферической крови крыс в 2-3 раза увеличилось количество лейкоцитов и в 1,6-1,75 раза возросло число ретикулоцитов. В костном мозге увеличилось количество пролиферирующих миелоидных и лимфоидных элементов, а также общее число клеток эритроидного ряда. Ксеногенная суммарная РНК стимулировала образование ЭО как на основе контакта свободных костномозговых макрофагов с молодыми эритроидными клетками (ЭО 1 и 2 классов), так и на базе реконструкции (ЭО реконструирующиеся). Сравнительный анализ эффектов нативной и лиофилизированной суммарной РНК не выявил различий между гемопоэтическими показателями у крыс, получивших эти препаратов. Заключение. Суммарная РНК, выделенная из лимфоидных клеток бычьей селезенки, активирует гемопоэз у крыс с постлучевой миелосупрессией, что свидетельствует об отсутствии ксеногенных ограничений у млекопитающих в механизмах лимфоцитарного контроля восстановительных процессов. Лиофилизированная суммарная РНК активирует костномозговое кроветворение в те же сроки и в том же объеме, что и нативная форма. Introduction. Allogeneic total RNA isolated from cells of lymphoid organs stimulates regeneration of hematopoietic tissue after acute and chronic disturbance of hematopoietic function. Aim. 1) To prove the absence of xenogeneic limitation for the lymphocytic regulation of regenerative processes using an example of the hemo-stimulating effect of total RNA from bovine spleen lymphocytes on hematopoiesis in rats exposed to sublethal gamma-irradiation; 2) To perform a comparative analysis of the effectiveness of the native and lyophilized forms of the total RNA. Methods. Experiments were performed on white outbred male rats weighing 200-220 g. Total RNA was isolated from bovine spleen lymphoid cells by phenol-chloroform extraction. To create the initial myelosuppression, 30 rats were exposed to a single general 60Co gamma radiation (6 Gy at 0.1 Gy/s). The rats were then divided into 3 equal groups. Two hrs after irradiation, the rats of the control group were injected intraperitoneally with 0.5 ml of 0.9% NaCl; rats of the second group received native total RNA, 30 μg/100 g body weight, and rats of the third group received lyophilized total RNA at a similar dose. On days 3, 7, and 12, the number of peripheral blood reticulocytes, leukocytes, and platelets was measured. The rats were then sacrificed, and bone marrow hematopoiesis was studied. After 12 days, the number of bone marrow erythroid, lymphoid, megakaryocytic, and myeloid cells was measured. Erythroblastic islets (EIs) were isolated from the bone marrow and differentiated into proliferating (class 1 and 2 EIs and reconstructing EIs) and mature (class 3 EIs and involving EIs) morpho-functional cell associations. Results. Under the influence of xenogeneic total RNA, the number of peripheral blood leukocytes increased by 2-3 times, and the number of reticulocytes increased by 1.6-1.75 times. In the bone marrow, the number of proliferating myeloid and lymphoid cells increased, as did the total number of erythroid cells. Xenogeneic total RNA stimulated formation of EIs, based both on the contact of free bone marrow macrophages with young erythroid cells (class 1 and 2 EIs) and on reconstruction (reconstructing EIs). Comparative analysis of the effects of native and lyophilized total RNA did not reveal differences between hematopoietic parameters in rats that received these agents. Conclusion. Total RNA isolated from bovine spleen lymphoid cells activates hematopoiesis in rats with post-radiation myelosuppression. This indicates the absence of mammalian xenogenic limitation of lymphocytic control of recovery processes. Lyophilized total RNA activates bone marrow hematopoiesis at the same rate and to the same extent as the native form.

Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors

Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta–gonad–mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.

Potent but transient immunosuppression of T-cells is a general feature of CD71+ erythroid cells

CD71+ erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-induced L-arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-γ production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-γ production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71highCD235adim cells). The suppressive properties disappear during erythroid differentiation as more differentiated CECs and mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of CECs in the immune response regulation.

LSD1 inhibition enhances robust fetal hemoglobin induction in human β0-thalassemia/HbE erythroid cells

Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of β-thalassemias. Histone methyltransferase LSD1 enzyme removes methyl groups from the activating chromatin mark histone 3 lysine 4 at silenced genes, including the γ-globin genes. LSD1 inhibitor RN-1 induces HbF levels in cultured human erythroid cells. Here, the HbF-inducing activity of RN-1 was investigated in erythroid progenitor cells derived from β0-thalassemia/HbE patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment was demonstrated in erythroid cells with divergent HbF baseline levels, the average of HbF induction was 17.7 + 0.8%. RN-1 at low concentration did not affect viability and proliferation of erythroid cells, but decreases in cell number was observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in β0-thalassemia/HbE erythroid cells treated with RN-1 as similar to other compounds that target LSD1 activity. Downregulation of repressors of γ-globin expression; NCOR1 and SOX6, was observed in RN-1 treatment. These findings provide a proof of concept that a LSD1 epigenetic enzymes is a potential therapeutic target for β0-thalassemia/HbE patients.

CD71+erythroid cells expansion in adult sepsis: potential causes and role in prognosis and nosocomial infection prediction

BackgroundImmune suppression contributes to nosocomial infections (NI) and poor prognosis in sepsis. Recent studies revealed CD71+ erythroid cells had unappreciated immunosuppressive functions. This study aimed to investigate the values of CD71+ erythroid cells in predicting NIs and prognosis among adult septic patients. The potential factors associated with the expansion of CECs were also explored.MethodsIn total, 112 septic patients and 32 critically ill controls were enrolled. The frequencies of CD71+cells, CD71+CD235a+ cells (CECs) and CD45+ CECs were measured by flow cytometry. The associations between CECs and NIs and 30-day mortality were assessed by ROC curve analysis, Cox and competing-risk regression model. Factors associated with the frequency of CECs were identified by linear regression analysis.ResultsThe percentage of CD71+cells, CECs, CD45+CECs were higher in septic patients than critically ill controls. In septic patients, the percentage of CD71+cells, CECs and CD45+CECs were associated with NI development, while CD71+cells and CECs were independently associated with 30-day mortality. Linear regression analysis showed that the levels of interleukin (IL)-6 and interferon (IFN)-γwere positively associated with the frequencies of CD71+cells, CECs and CD45+CECs, while IL-10 was negatively associated with them. Additionally, the levels of red blood cells (RBCs) were negatively associated with the percentage of CD45+ CECs.ConclusionsCECs were expanded in sepsis and can serve as independent predictors of the development of NI and 30-day mortality. Low levels of RBCs and high levels of IL-6 and IFN-γmay contribute to the expansion of CECs in sepsis.Trial registration: ChiCTR, ChiCTR1900024887. Registered 2 Augest 2019, http://www.chictr.org.cn/showproj.aspx?proj=38645

Proteome Expression Profile for Red Blood Cells Enables Diagnostics for Hepatocellular Carcinoma

BackgroudEarly diagnosis of hepatocellular carcinoma (HCC) has not been clinically resolved, which has been causing more death in patients with HCC. HCC is also a systemic disease related to disorders of blood homeostasis, and the association of red blood cells (RBCs) and HCC tumorigenesis is still elusive. This study explored the protein characteristics of RBCs at the progressive pathological stages comparing with healthy individuals, including liver cirrhosis (LC) and established HCC, to fully understand the tumorigenesis of HCC from a different view and identify potentially novel diagnostic biomarkers for HCC in RBCs.MethodsData independent acquisition (DIA) proteomic analyses were performed with 72 clinical RBCs samples from a cohort of subjects including HCC, LC and healthy controls. Bioinformatics analysis was conducted for significantly differentially expressed proteins (DEPs) through the whole process of tumorigenesis to characterize the clinical relevanve of RBCs and tumorigenesis in HCC. The highly potential tumorigenesis-associated molecular biomarkers were evaluated with clinical samples by parallel reaction monitoring (PRM) technology.ResultsWe observed that red blood cells number dynamically changes during the tumorigenesis of HCC, and LC is a developmental stage more closely approaching HCC based on the protein expresson profiles in RBCs. The expression of hemoglobin (HbA, HbF) in erythroid cells also dynamically alters during the whole process of HCC tumorigenesis, suggesting immature erythroid cells exist in peripheral blood of HCC patients and erythropoiesis in the patients starts to be influenced with the occurance of LC. We observed that the autophagy pathway is disturbed in RBCs with the onset of LC and maintained during the tumorigenesis of HCC. Oxytocin pathway and GnRH pathway are disturbed and first identified during the development of LC into HCC. SMIM1, ANXA7, HBA1 and HBE1 that are significantly altered during tumorigenesis were verified as promising biomarkers for HCC early diagnosis.ConclusionsThis study underlied the clinical relevance of the proteins in RBCs and the tumorigenesis of HCC, and provided the potential biomarkers for early diagnosis in HCC from a new perspective. Our results provided a novel strategy with RBCs for HCC early diagnosis, which will improve the translational research and application in diagnosis of HCC.

The Condensin II Complex Subunit NCAPH2 Is Essential for Erythropoiesis and Regulates Erythroid Gene Expression

Erythropoiesis requires dramatic changes in gene expression in a cell that is rapidly proliferating and undergoing progressive nuclear condensation in anticipation of enucleation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Our lab has demonstrated that Setd8, the sole histone methyltransferase that can generate H4K20me1, plays an essential role in this process (Malik 2019). H4K20me1 accumulates during terminal erythroid maturation (Murphy Blood 2021) and can regulate chromatin structure and gene expression through interaction with multiple partners, including the Condensin II Complex. The Condensin II complex is a ring-like structure composed of two conserved SMC components (SMC2 and SMC4), two HEAT subunits (NCAPG2 and NCAPD3), and a kleisin subunit NCAPH2. The Condensin II complex plays an important role in chromatin condensation during mitosis, and establishing higher-order chromatin interactions in interphase cells, with some studies suggesting it also regulates gene expression (Yuen Science Adv 2017; Iwasaki Nature Comm 2019). Similar to Setd8, many subunits of the Condensin II complex are highly expressed in erythroid cells compared to most other cell types (biogps.org). We hypothesized that the Condensin II complex, in conjunction with Setd8 and H4K20me1, is important for establishing appropriate patterns of chromatin architecture and gene expression in maturing erythroblasts. To address this hypothesis, we deleted the NCAPH2 subunit in erythroid cells by crossing mice with floxed alleles of NCAPH2 with mice expressing cre-recombinase under the direction of the Erythropoietin receptor promotor (EpoRCre). Homozygous disruption of NCAPH2 was embryonic lethal by E13.5. NCAPH2 mutant embryos were similar in appearance to littermate controls until E12.5 when they developed notable pallor and a dramatic decline in the number of benzidine positive cells. Cell cycle analyses demonstrated that an accumulation of cells in G2/M preceded the dramatic decline in erythroblast numbers at E12.5. In contrast to cells from littermate controls, the NCAPH2 mutant cells were very heterogenous in cell and nuclear size and morphology. Surpisingly, most NCAPH2 mutant cells appeared to be hemoglobinized, suggesting sufficient iron accumulation and heme synthesis. In vitro cultures derived from primitive erythroid progenitors replicated in vivo findings, including normal early erythropoeisis, with significant abnormalities during mid- to late- maturation. Western blot in cycloheximide treated primitive erythroid cultures revealed that NCAPH2 has a long half-life, which likely contributes to the relative normalicy of early primitive erythoproesis. NCAPH2 mutant embryos also had a dramatic failure of definitive erythropoiesis, as evidenced by loss of mature erythroblasts in the fetal liver at E13.5. To gain insights into the mechanisms underlying these findings, we performed RNA-seq of NCAPH2 mutant, NCAPH2 het, and NCAPH2 WT erythroblasts from E11.5 embryos. Comparing NCAPH2 mutant and NCAPH2 WT erythroblasts there were 1121 down regulated genes and 743 upregulated genes (adj p-value <0.05). As expected, the downregulated genes were enriched for pathways related to cell cycle, such as Mitotic Spindle Organization (adj pvalue 5e-42). The upregulated genes were enriched for a variety of pathways including p53 transcriptional network (adj pvalue 4e-10), neutrophil mediated immunity (2e-9), DNA-binding transcription factor (adj pvalue 7e-5), and regulation of erythrocyte differentiation (adj pvalue 5e-4). Intriguingly, 91/340 genes differentially expressed in Setd8 mutant erythroblats were also differentially expressed in NCAPH2 mutant cells, including genes typically repressed early in erythroid commitment, such as GATA2 and SPI1. Cut&Tag in CD34+ derived erythroblasts demonstrated occupancy of H4K20me1 at these loci. Mass spectrometry of proteins isolated via mono-mehtylated H4K20 peptides in erythroid extracts identified Condensin II components, supporting a model where the Condensin II complex directly interacts with H4K20me1. Together, these results demonstrate that the Condensin II complex is essential for erythropoiesis, and may work in conjunction with Setd8 and H4K20me1 to establish appropriate patterns of gene expression in maturing erythroblasts. Disclosures No relevant conflicts of interest to declare.

Protein Phosphatase 6C (PPP6C) Loss Significantly Raises Fetal Hemoglobin Levels and Reduces Cell Sickling

Sickle cell disease (SCD) afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has remained an elusive goal. To identify additional potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen targeting all protein phosphatases (1308 independent sgRNA representing 218 phosphatases). The phosphatase sgRNA library was cloned into a lentivirus scaffold and introduced into the erythroid cell line HUDEP2 stably expressing Cas9, and the top and bottom 10% of HbF-expressing cells were sorted and the integrated sgRNAs were sequenced. This screen identified a single protein phosphatase - PPP6C - as an HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6, a serine/threonine cytosolic protein phosphatase that is widely expressed across tissues and throughout erythroid development to broadly regulate mRNA translation. PPP6C has been implicated in numerous cellular functions, including cell cycle regulation, autophagy, and innate immunity, but its role in HbF regulation has not previously been described. Depletion of PPP6C by 5 independent sgRNAs in HUDEP2 cells resulted in significant HbF enrichment. Importantly, PPP6C depletion did not affect cellular viability or differentiation, suggesting that PPP6C may serve as a targetable HbF regulator for the treatment of SCD. To validate the findings of this genetic screen in primary human erythroid cells, we performed CRISPR-Cas9 ribonuclear protein (RNP)-based genome editing of PPP6C in a three-phase in vitro culture of adult CD34+ hematopoietic cells. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We find that depletion of PPP6C protein levels by greater than 80% increases gamma-globin transcript levels in a dose-dependent manner to nearly 5 times basal levels. In addition, PPP6C loss leads to a greater than doubling in F-cell number and a 3-4-fold increase in HbF levels as measured by HPLC analysis. PPP6C depletion showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Mechanistically, loss of PPP6C leads to depletion of BCL11A protein levels by nearly 50% but unchanged levels of other key HbF regulators such as HRI and LRF, suggesting PPP6C-mediated HbF regulation may proceed at least in part via loss of BCL11A. However, additional studies are necessary to fully elucidate these underlying regulatory mechanisms. Importantly, depletion of PPP6C in SCD patient-derived cells was well tolerated, led to similar levels of HbF induction, and markedly reduced cell sickling by greater than 60%. Results from ongoing studies exploring the mechanism of PPP6C in HbF regulation will be discussed. Taken together, these data indicate that PPP6C functions in a dose-dependent manner to regulate HbF in primary erythroid cells and may serve as a therapeutic target in the treatment of SCD. Disclosures Blobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

Multicenter Prospective Evaluation of Diagnostic Potential of Flow Cytometric Aberrancies in Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) are considered clonal diseases and are diagnosed according to WHO by cytomorphology and cytogenetics. The diagnostic potential of flow cytometric aberrancies has not yet been comprehensively evaluated. Aim: Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies predefined according to European LeukemiaNet (ELN). Methods: 1682 patients undergoing diagnostics for suspected MDS according to WHO 2016 criteria were analyzed in parallel by flow cytometry according to ELN recommendations. Results: Median age was 72 years (18-97). MDS, MPN-RS-T or CMML were confirmed by cytomophology in 1029 (61%) cases, 653 (39%) were non-MDS. IPSS-R data was available in 857 (51%). An overall flow cytometric readout was available in 1679 (99.8%). 1001 (60%) were in agreement with MDS while 678 (40%) were not. Flow cytometric readout significantly correlated with cytomorphologic diagnosis (p<0.001): 850 (51%) were positive by both methods (flow+/cyto+), 502 (30%) were flow-/cyto-, 176 (10%) were flow-/cyto+ and 151 (9%) flow+/cyto-. The rate of flow+ was higher in high-risk MDS (MDS-EB1/2, 92%) and CMML (89%) compared to low-risk MDS (76%). Accordingly, regarding IPSS-R highest frequency of flow+ was found in very high risk (96%) and lowest one in very low risk group (64%). Non-MDS cases had a fewer myeloid progenitor cells (MPC) (mean±SD, 0.8±0.9%) compared to low-risk MDS (1.7±2.3%, p<0.001), high-risk MDS (6.3±5.0%, p<0.001) and CMML (1.9±2.6%, p<0.001). In particular, MPC >3% was strongly associated with MDS/CMML (286/293, 98%, p<0.001). Antigen expression aberrancies in MPC were more frequent in cases with MDS or CMML than in non-MDS cases but differed between entities with lower frequencies in low-risk MDS cases (table 1). Neutrophil aberrancies were found more frequently in neoplastic cases than in non-MDS cases (table 1). Again, frequencies of aberrations were higher for high-risk MDS as compared to low-risk MDS while this was not the case for CMML showing frequencies rather similar to low-risk MDS. Frequencies of aberrancies in monocytes revealed a similar figure as in neutrophils with higher rates in neoplastic cases but clearly significant numbers positive in non-MDS cases. Interestingly, frequencies were not higher in high-risk MDS as compared to low-risk MDS. As anticipated, frequencies were highest in CMML (table 1). Regarding erythroid cells only an aberrant percentage of them and aberrant CD71 expression were found in a reasonable number of cases. Importantly, rates of positivity were rather high in non-MDS cases which did not differ from CMML cases (table 1). In order to identify the diagnostic value of each individual aberrancy multivariate analyses were performed in the three subgroups, low-risk MDS, high-risk MDS and CMML, as well as in the total cohort. In low-risk MDS ten aberrancies were independently related to MDS (table 2). Five of these aberrancies were found in MPCs, two each in neutrophils and monocytes and one in erythroid cells. In high-risk MDS 11 aberrancies were independently related to MDS (table 2). Eight were found in MPCs, two in neutrophils, none in monocytes and one in erythroid cells. In CMML 12 aberrancies were independently related to CMML (table 2). Four were found in MPCs, neutrophils and monocytes, respectively, and none in erythroid cells. Considering all these three groups together and all aberrancies identified significantly related to MDS/CMML in at least one group in univariate analysis, multivariate analysis identified 12 aberrancies independently related to MDS/CMML (table 2). Six were found in MPCs, two in neutrophils, three in monocytes and one in erythroid cells. Taking into consideration only aberrancies independently associated with MDS/CMML, three such aberrancies resulted in an 80% agreement with the cytomorphologic diagnosis of MDS/CMML, i.e. 20% concordantly negative and 60% concordantly positive. Importantly, this applies without need of at least two cell compartments being affected as specified in the ELN recommendations. Conclusions: This multicenter prospective evaluation confirms the diagnostic potential of flow cytometric aberrancies. A core set of 17 markers identified as independently related to a diagnosis of MDS/CMML is suggested mandatory for flow cytometric evaluation of suspected MDS. An MPC count >3% should be considered indicative of MDS/CMML. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Van de Loosdrecht: Celgene: Consultancy, Research Funding; Amgen: Consultancy; Roche: Consultancy; Novartis: Consultancy; Alexion: Consultancy.